Comparison of Osteoblastic Differentiation of Human Periodontal Ligament Stem Cells through Application of Two β-tricalcium Phosphate Products: An in vitro Study

Statement of the Problem: Osteoblastic differentiation of periodontal ligament stem cells (PLSCs) is essential for alveolar bone regeneration. Purpose: The purpose of this study was to compare the potential of two β-tricalcium phosphate (βTCP) products to induce osteoblastic differentiation of human PLSCs. Materials and Method: In this in vitro study, human PLSCs were cultured in mediums supplemented with Guidor Easy-Graft [βTCP+polylactide-co-glycolide (PLCG)+n-methyl-2-pyrrolidone (NMP)] [Sunstar Company, Swiss] or Sorbone [βTCP] [Meta Company, South Korea] as two alloplasts experimental groups, mesenchymal cells differentiated into osteoblasts without alloplast as positive control group, and mesenchymal cells without osteoblastic induction as negative control group. Osteoblastic differentiation was evaluated using Alizarine Red staining and spectrophotometry to assay calcium deposits and real-time polymerase chain reaction to examine expression of alkaline phosphatase (ALP) and osteopontin (OPN) antigens on day 21. Data were analyzed by using SPSS 22 software and one-way ANOVA and Bonferoni tests (p< 0.05). Results: Spectrophotometry confirmed that calcium deposits were higher in Guidor Easy-Graft group compared to Sorbone group (p< 0.001) and higher in two experimental groups than controls (p< 0.05). According to real-time polymerase chain reaction, level of ALP expression was higher in Sorbone than Guidor and the levels of Guidor, positive control and negative control were equal; OPN levels of the positive control were more than the other groups. OPN levels of Sobone, Guidor and negative control were the same. Conclusion: These findings indicated that Guidor Easy-Graft and Sorbone enhanced differentiation of human PLSCs to osteoblasts, and could be employed as appropriate bone-graft materials.


Introduction
In clinical dentistry, bone regeneration is used for treatment of alveolar bone resorption, ridge or socket preservation, preparation for dental implant placement and maxillary sinus floor augmentation [1]. Natural (autografts, allografts, and xenografts) or synthetic (alloplasts) materials implement bone grafting, as an approach to bone regeneration. Alloplasts including calcium phos-phates (tricalcium phosphate, hydroxyapatite, and calcium polyphosphates), bioactive glasses, and calcium sulfate were introduced to overcome the natural material disadvantages. Disadvantages of autografts are related to limited intraoral access to the donor area as well as the amount of the tissue obtained [2]. Allografts and Xenografts may also lead to disease transmission or immune stimulation [3]. Alloplasts are synthetic, inorganic, and biocompatible materials used as bone substitutes and fillers for intraosseous defects, which can support bone regeneration during the osteoconduction process [4].
Two commercial beta-tricalcium phosphate (β-TCP) products include Guidor Easy-Graft and Sorbone. Guidor consists of a powder containing β-TCP particles, a thin polymer coating poly lactide-co-glycolide (PLCG) and an activating liquid n-methyl-2-pyrrolidone (NMP) commercially named BioLinker. Sorbone contains pure beta-tricalcium phosphate [5][6]. Two previous studies have shown the capability to induce new bone formation by β-TCP exclusively [5] or in combination with PLCG [6]. PLCG can assist the formation of new bone due to calcium releasing capability [7] and also facilitates β-TCP application, increases its stiffness and stability, as well as improves its osteoconductivity and porosity as a scaffold [8].
Periodontal ligament stem cells (PLSCs) are subset of mesenchymal stem cells that can be easily extracted from periodontal tissue. These cells have a high ability to form new bone, cement, and periodontal ligament [9] and can differentiate into chondrocytes, osteoblasts, fibroblasts, and adipocytes under special conditions [10][11]. In particular, the ability of PLSCs to differentiate into osteoblasts has made them an appropriate candidate for the treatment of bone defects [12]. Osteoblastic differentiation of these cells could be confirmed by detection of differentiation markers including alkaline phosphatase (ALP) activity, mineralization capability, and expression of osteoblastic genes encoding ALP, osteopontin (OPN), osteocalcin, and bone sialoprotein [13].
Other methods including ALP staining [14], Alizarin Red staining for detection of tissue calcium [15] and polymerase chain reaction for determination of expression of different genes [16] are also performed to confirm osteoblastic differentiation. Stem cell differentiation is highly dependent on cell interaction with the physical, mechanical, and biological properties of the growth environment [8]. To evaluate the differentiation into osteoblasts, PLSCs of the fourth passage were cultured in Dulbecco's Modified Eagle Medium (medium containing 10% bovine serum, dexamethasone, 0.2mM ascorbic acid, and 10mM beta-glycerophosphate) to induce bone differentiation. The cells were transferred to culture flasks at a concentration of one million cells per milliliter and they were incubated under normal cell culture conditions. After 48 hours, the flask culture medium was changed and then the cell culture medium was changed every three days.
When the cell density reached 80%, cell passage took place. As a negative control group, the cells were cultured in Dulbecco's Modified Eagle Medium containing 10% bovine serum. The cells were then incubated at 37°C and 5% CO2 for 21 days. Their medium was changed every two days. At the end of this period, the occurrence of cell differentiation was explored by Alizarin Red staining. Thereby, the cell monolayer was washed with phosphate buffered saline and fixed by pure methanol for 10 min, then stained with 2% Alizarin Red dye in 25% ammonia water for 2 min. Finally, the cells were washed with distilled water, dried, and then examined by a microscope.
Using the "ELISA Reader" spectrophotometer, the quantity of 405 nm light absorption was measured as a marker for the amount of calcium deposits. The expression of ALP and OPN genes, which indicate differentiation into osteoblast cells, was measured by polymerase chain reaction technique in all groups. In our study, osteoblastic differentiation of PLSCs was assessed by Alizarin Red staining and polymerase chain reaction on day 21, the time that required for differentiation of these cells [17]. Data were analyzed by SPSS 22 software using one-way ANOVA and Bonferoni tests (p< 0.05).  Table 1).

Microscopic evaluation of
The mean expression of ALP gene in Sorbone group was significantly higher than Guidor group (p= 0.005), negative and positive controls (p= 0.001). There was no   and negative control groups (p> 0.05) ( Table 2).

Discussion
Alloplasts have been known as a practical option to treat bone defects due to periodontal diseases. β-TCP materials are very common practical alloplasts. β-TCP has a crystalline structure and its porosity provides desirable biological properties such as osteoconduction and resorption. It also causes the formation of new bone within 3 to 6 months by releasing calcium and phosphate ions [18]. The effects of β-TCP in pure form or in combination with other substances on the differentiation of PLSCs into other cell lines have been previously studied [19][20].
In the present study, human PLSCs were used be- Finally, they decrease inflammatory reactions and immune responses [21].
In our study, Alizarin Red staining/ spectrophotometry and expression of ALP and OPN genes were used to evaluate osteoblastic differentiation. Alizarin Red staini- hence, identifies the osteoblastic phenotype. In addition, the expression of ALP and OPN genes are used to identify genes associated with this phenotype [22].
In this study, four groups including two experimental groups (Guidor and Sorbone groups), one positive control group, and one negative control group were evaluated. Evaluation of osteoblast differentiation by spectrophotometry showed that calcium deposition was negligible in the negative control group. Therefore, the increase in calcium deposition in the other three groups can be attributed to its production by osteoblasts differentiated from the stem cells. two previous studies [23][24] have shown that calcium phosphate could increase bone formation, osteoblast proliferation, and activity. In a clinical study, Naineni et al. [25] found that the usage of β-TCP-containing materials in patients with alveolar bone defects inhibited alveolar crest degeneration and enhanced bone formation. Amalakara et al. [26] reported that β-TCP alone or in combination with an immunosuppressive agent (cyclosporine A) improved alveolar bone growth and caused filling of bone defects. According to a clinical trial by Cochran et al. [27], periodontal bone defects were treated by β-TCP/rh-FGF-2. Maroo and Murthy [28] evaluated patients with periodontal intraosseous defects and reported the efficacy of β-TCP/rhPDGF combination in filling bone defects and increasing alveolar margin height.
Calcium deposition was significantly higher in Guidor group compared to Sorbone group. Although the main component of both products is β-TCP, there are differences in composition and structure between two products. Guidor contains PLCG that is activating by liquid (NMP). It has been shown that PLCG supports the formation of new bone by releasing calcium [7].
Therefore, the higher calcium deposition in Guidor group than Sorbone group can be attributed to the presence of PLCG in Guidor.
The mean expression of ALP gene in Sorbone group was significantly higher than the positive control. This finding indicates that the expression of ALP gene as a marker of osteoblastic differentiation increased under the influence of Sorbone. A study on animal cells showed that incubation of bone marrow stromal cells in the presence of β-TCP significantly increased ALP production [5]. McCafferty et al. [29] observed that the addition of calcium phosphate coating on titanium surfaces improved the osteogenic differentiation of mesenchymal stem cells cultured on these surfaces. According to An et al. study [19], culturing human PLSCs in the presence of β-TCP, increased the ALP activity. Xia et al. [20],investigated the osteogenic differentiation of human PLSCs based on the expression of ALP and OPN genes and reported that the expression of the genes was lower in β-TCP group than acermanite group.
In addition, An et al. [19], observed that during osteo- showed some degree of inhibitory effect on ALP gene expression but it could play a role in improving calcification. Guidor Easy-Graft and Sorbone did not differ significantly in terms of OPN gene expression.